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* These authors contributed equally
This protocol describes an efficient, simple, and minimally invasive method for studying pulmonary nodules. Submaxillary vein blood collection and micro-CT imaging are used as investigative techniques.
Micro-computed tomography (micro-CT) is a real-time, intuitive, sensitive, and minimally invasive technique for monitoring changes from pulmonary nodules (PN) to lung cancer (LC). The integration of submandibular vein blood sampling enables rapid, stable, and straightforward detection of imaging and key target alterations during the progression of PN to LC. In this study, we administered a dosage of 100 mg/kg of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice to develop a lung adenocarcinoma model. Disease progression in the experimental animals was then monitored through submandibular vein blood sampling and micro-CT assay. Experimental results showed the presence of nodular foci in the lungs of some animals by the 10th week, with the development of lung adenocarcinoma images becoming evident by the 21st week. In conclusion, micro-CT can effectively observe pathological changes in the lungs of mice and, when combined with submandibular vein blood sampling, can dynamically monitor changes in blood, protein, and targets. This method provides a highly specific, simple, and sensitive approach for drug screening, pharmacokinetic testing, toxicological experiments, and safety studies.
Lung cancer (LC) is a severe neoplasm originating in the bronchial mucosa or lung glands. According to 2021 statistics, LC causes approximately two million fatalities globally each year, with incidence and mortality rates on the rise1. Early diagnosis and intervention in LC contribute to higher cure rates, reduced mortality, and lower treatment costs. Pulmonary nodules (PN) are specific precursors to LC, characterized by localized, round, and denser solid or subsolid shadows ≤30 mm in diameter on radiological exams, without evidence of lung collapse, mediastinal lymph node enlargement, or pleural effusion2. The National Comprehensive Cancer Network (NCCN) in 2022 categorized PN by number, diameter, and density, identifying combinations such as a 5 mm isolated ground-glass nodule in the right lung3. However, the NCCN guidelines indicate that the risk of malignancy in PN increases with the nodules' diameter and quantity. The widespread application of low-dose computed tomography has dramatically increased PN diagnoses, with millions of new cases identified each year4.
The combination of A/J mice with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most commonly used animal model for lung cancer (LC)5,6. The use of micro-CT alongside submandibular vein blood sampling is an effective approach for real-time monitoring of changes from pulmonary nodules (PN) to LC. Chemical carcinogen induction, particularly with NNK and A/J mice, is the most prevalent method for lung cancer modeling and has proven to be an efficacious approach for establishing carcinoma in situ7,8. This modeling method more accurately simulates the progression of PN to LC compared to the axillary inoculation method.
Previous studies have focused on statistical analysis of nodule morphology and pathological staining of tissue samples post-euthanasia9. However, these methods lack the capacity for real-time monitoring of the dynamic progression from PN to LC10. Micro-CT, as a non-invasive imaging technique, provides accurate longitudinal data with high resolution, fast imaging, a low radiation dose, and safety, making it suitable for detecting lung images in real time11,12. Submandibular vein blood sampling is the latest, simplest, and fastest method for obtaining blood samples from mice13. This non-invasive technique requires minimal animal handling and allows for rapid recovery, aligning with the 3R principles that aim to reduce the number of animals used in research, minimize discomfort, and promote ethical treatment. The collected blood volume, approximately 0.2-0.5 mL, is sufficient for monitoring blood parameters with moderate requirements14.
The concurrent use of micro-CT and submandibular vein blood sampling allows for dynamic, real-time observation of PN-to-LC progression in imaging and the real-time detection of key targets within the bloodstream15. Additionally, this approach enables real-time investigation of metabolites and other biochemicals, which, when combined with techniques such as high-performance chromatography, advances our understanding of LC16,17.
In this study, A/J mice combined with NNK were used to create an in-situ lung cancer mouse model. Micro-CT scans were performed at 4, 10, and 20 weeks post-model induction to capture lung images, while blood was collected via submandibular vein sampling throughout the experiment. This study aims to establish a foundation for PN and LC research by combining submandibular vein blood sampling with micro-CT.
In oncology, micro-CT is a highly effective tool for detecting tumor growth, offering a high-resolution technique for measuring local shadow-focus changes at any time during such studies18,19. However, it is essential to recognize that micro-CT alone does not provide information on shadow-focus characteristics, the physiological status of the animal, or levels of key biological factors. Therefore, submandibular vein sampling was utilized as a complementary method in this study.
All animal experiments described in this study were approved by the Experimental Animal Welfare Ethics Committee of Chengdu University of Traditional Chinese Medicine and were conducted in accordance with relevant laws and ethical standards for animal research (review number: 2024035). Female inbred A/JGpt mice (7-8 weeks old) were maintained at a temperature of 20-24 °C with a relative humidity of 40%-70%. They were provided with standard animal feed and purified water ad libitum throughout a 12-h light-dark cycle. Before the experiment, each animal was acclimated to this environment for 7 days. Details of the reagents and equipment used are listed in the Table of Materials.
1. Reagents and animal preparation
2. In vivo imaging by micro-CT
NOTE: Always remove metal objects, such as ear tags, from the test animal before using the micro-CT scan. Metal objects can cause severe artifacts in the image. Micro-CT emits a certain amount of radiation; ensure that other experimental results are not affected.
3. Data processing and analysis
This study demonstrated the construction of a stable lung cancer model using NNK in combination with A/J mice. The experimental design is illustrated in Figure 1. The objective was to observe the real-time process of the transition from pulmonary nodules (PN) to lung cancer (LC) in mouse lungs, utilizing micro-CT and submandibular vein blood sampling. Accordingly, micro-CT scanning and blood sampling from the mouse lungs were conducted at the fourth, tenth, and twentieth weeks.
It is important to reiterate several key points from this study. Firstly, although submandibular vein blood collection is a relatively low-injury procedure, it may still result in some degree of harm to the animals. Therefore, it is necessary to conduct multiple procedures to reduce the burden on the mice and complete the process in a timely manner27. Secondly, the removal of hair prior to blood sample collection ensures the purity of the sample. Thirdly, it is imperative to use appropriate blood ...
The authors have nothing to disclose.
We thank Professor Cong Huang of the School of Basic Medical Sciences and Professor Yan Huang of the School of Pharmacy, Chengdu University of Traditional Chinese Medicine, for their support. We would also like to thank Dr. Binjie Xu and Dr. Pengmei Guo. (Innovative Institute of Chinese Medicine and Pharmacy, Chengdu
University of Traditional Chinese Medicine) for providing instrument and Technical support.
Name | Company | Catalog Number | Comments |
A/J mice | GemPharmatech LLC. | N000018 | |
0.5 mL EDTA tubes | Labshark | 130201070 | |
1-Butanone,4-(methylnitrosoamino)-1-(3-pyridinyl) | Gu Shi Gong Yuan Medical Equipment Co. | N589770 | |
75% ethanol | ChengDu Chron Chemicals Co,.Ltd | 2023052901 | |
Animal shaver | Codos | BM010220 | |
Isoflurane | Shenzhen Reward Life Technology Co. | R510-22-16 | |
medical tricorder | MedChemexpress | 69652 | |
Quantum GX2 microCT imaging system | PerkinElme | 2020166501 | |
Saline (medicine) | Beijing Biolabs Technology Co. | GL1736 |
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